Before we explain what a Ct (Cycle Threshold) value is, let’s go back to the basics of PCR for the use of diagnosis.
PCR stands for Polymerase Chain Reaction. There is cyclic amplification of DNA through the use of specific enzymes. This amplification follows an exponential or chain reaction pattern. In the case of RNA viruses, the RNA in the sample is converted to the complementary DNA using a reverse transcriptase enzyme (hence the name RT-PCR: Reverse Transcriptase Polymerase Chain Reaction).
Without going into too much detail, what happens is the following:
We are now in a position to define what a Ct value is. The Ct (cycle threshold) is defined as the number of cycles (of amplification) required for the fluorescent signal to cross the threshold (i.e exceeds background level) in a positive sample. So, in a negative sample, no Ct value is generated.(1)
Theoretically, Ct levels are inversely proportional to the amount of target nucleic acid in the sample.(1)
Presently, qualitative kits are being used for detection of the SARS-CoV-2 virus. These kits are not validated for quantitation of the viral load. That will only be possible with kits specifically developed and validated for measuring the viral load in a given sample.
Not really. Even though the Ct value may theoretically correspond with viral load, there are too many pitfalls associated with using Ct value to guide treatment.
The pitfalls that may occur in using Ct value to interpret viral load belong to four major categories
Shedding of the virus in the respiratory tract will be influenced by co-morbidities (either diagnosed or undiagnosed), or genetic factors (which may or may not predispose to a more severe disease course). It is unknown at this point whether the amount of virus in an individual or the amount of virus shed by the individual, plays any additional role in this context.(2)
Viral factors may also influence the interpretation of Ct values.(2) The SARS-CoV-2 virus has showngreatvariations in its shedding pattern in an infected individual. Cases have been reported where there was prolonged virus shedding even after the clinical symptoms were resolved and the individual developed antibodies.(3) We still require more long term studies and data to be able to interpret the shedding pattern and duration of shedding of this virus.
Ct values can vary due to a variety of factors including site of swab collection, type of local immunity& how the sample is collected and transported. This is why the Ct value can vary from sample to sample even in the same individual.One paper on acute respiratory illness stated that, in an adult patient, they found 1000-fold difference in human DNA concentration between two broncho-alveolar lavage samples, taken by the same pulmonologist, potentially reflecting a similar difference in viral sample size.(2)
The Ct value is determined by using a qualitative assay and does not correlate directly with viral load as none of the kits use quantify the same.Ct values for a specimen vary between different kits and techniques. The target genes differ from kit to kit. Primers for the same gene may also differ between kits. Also, to be noted is that Ct values are valid for the kit used and that test-run only. A re-run of the same specimen on the same kit may return a different Ct value based on the assay dynamics. The efficiency of the first step of the RT-PCR procedure, nucleic acid extraction, may also vary between samples with different composition and nucleic acid concentrations. The efficiency of both steps may vary considerably between different target sequences. A given RNA or DNA concentration may result into different CT values depending on the target sequence, on the reverse-transcription and polymerase enzymes, and on the reaction conditions used.(2)
Only use viral load values from validated quantitative assays (when they become available) to guide treatments. Though Ct values may mirror the viral load in a particular sample, its limitations and inherent variability needs to be kept in mind before making clinical decisions based off it.